Chang Qing Cao

A peripherally restricted cannabinoid receptor agonist produces robust anti-nociceptive effects in rodent models of inflammatory and neuropathic pain.

&NA; Cannabinoids are analgesic in man, but their use is limited by their psychoactive properties. One way to avoid cannabinoid receptor subtype 1 (CB1R)‐mediated central side‐effects is to develop CB1R agonists with limited CNS penetration. Activation of peripheral CB1Rs has been proposed to be analgesic, but the relative contribution of peripheral CB1Rs to the analgesic effects of systemic cannabinoids remains unclear. Here we addressed this by exploring the analgesic properties and site of action of AZ11713908, a peripherally restricted CB1R agonist, in rodent pain models. Systemic administration of AZ11713908 produced robust efficacy in rat pain models, comparable to that produced by WIN 55, 212–2, a CNS‐penetrant, mixed CB1R and CB2R agonist, but AZ11713908 generated fewer CNS side‐effects than WIN 55, 212‐in a rat Irwin test. Since AZ11713908 is also a CB2R inverse agonist in rat and a partial CB2R agonist in mouse, we tested the specificity of the effects in CB1R and CB2R knock‐out (KO) mice. Analgesic effects produced by AZ11713908 in wild‐type mice with Freunds complete adjuvant‐induced inflammation of the tail were completely absent in CB1R KO mice, but fully preserved in CB2R KO mice. An in vivo electrophysiological assay showed that the major site of action of AZ11713908 was peripheral. Similarly, intraplantar AZ11713908 was also sufficient to induce robust analgesia. These results demonstrate that systemic administration of AZ11713908, produced robust analgesia in rodent pain models via peripheral CB1R. Peripherally restricted CB1R agonists provide an interesting novel approach to analgesic therapy for chronic pain.

Blocking spinal CCR2 with AZ889 reversed hyperalgesia in a model of neuropathic pain

BackgroundThe CCR2/CCL2 system has been identified as a regulator in the pathogenesis of neuropathy-induced pain. However, CCR2 target validation in analgesia and the mechanism underlying antinociception produced by CCR2 antagonists remains poorly understood. In this study, in vitro and in vivo pharmacological approaches using a novel CCR2 antagonist, AZ889, strengthened the hypothesis of a CCR2 contribution to neuropathic pain and provided confidence over the possibilities to treat neuropathic pain with CCR2 antagonists.ResultsWe provided evidence that dorsal root ganglia (DRG) cells harvested from CCI animals responded to stimulation by CCL2 with a concentration-dependent calcium rise involving PLC-dependent internal stores. This response was associated with an increase in evoked neuronal action potentials suggesting these cells were sensitive to CCR2 signalling. Importantly, treatment with AZ889 abolished CCL2-evoked excitation confirming that this activity is CCR2-mediated. Neuronal and non-neuronal cells in the spinal cord were also excited by CCL2 applications indicating an important role of spinal CCR2 in neuropathic pain. We next showed that in vivo spinal intrathecal injection of AZ889 produced dose-dependent analgesia in CCI rats. Additionally, application of AZ889 to the exposed spinal cord inhibited evoked neuronal activity and confirmed that CCR2-mediated analgesia involved predominantly the spinal cord. Furthermore, AZ889 abolished NMDA-dependent wind-up of spinal withdrawal reflex pathway in neuropathic animals giving insight into the spinal mechanism underlying the analgesic properties of AZ889.ConclusionsOverall, this study strengthens the important role of CCR2 in neuropathic pain and highlights feasibility that interfering on this mechanism at the spinal level with a selective antagonist can provide new analgesia opportunities.

Pro-nociceptive effects of neuromedin U in rat.

The neuropeptide neuromedin U (NMU) has been shown to have significant effects on cardiovascular, gastrointestinal and CNS functions. The peptide was first isolated from the porcine spinal cord and later shown to be present in spinal cords of other species. Little is known about the distribution of neuromedin U receptors (NMURs) in the spinal cord and the spinal action of the peptide. Here we report on the expression of NMURs and a potential role in nociception in the rat spinal cord using a combination of behavioral and electrophysiological studies. Receptor autoradiography showed that NMU-23 binding was restricted to the superficial layers of spinal cord, a region known to be involved in the control of nociception. In situ hybridization analysis indicated the mRNA of NMUR2 was located in the same region (laminae I and IIo) as NMU-23 binding, while the mRNA for NMU receptor 1 was observed in a subpopulation of small diameter neurons of dorsal root ganglia. Intrathecal (i.t.) administration of neuromedin U-23 (0.4-4.0 nmol/10 microl) dose-dependently decreased both the mechanical threshold to von Frey hair stimulation and the withdrawal latency to a noxious thermal stimulus. Mechanical allodynia was observed between 10 and 120 min, peaking at 30 min and heat hyperalgesia was observed 10-30 min after i.t. administration of NMU-23. A similar mechanical allodynia was also observed following i.t. administration of NMU-8 (0.4-4 nmol/10 microl). A significant enhancement of the excitability of flexor reflex was induced by intrathecal administration of NMU-23 (4 nmol/10 microl). Evoked responses to touch and pinch stimuli were increased by 439+/-94% and 188+/-36% (P<0.01, n=6) respectively. The behavioral and electrophysiological data demonstrate, for the first time, a pro-nociceptive action of NMU. The restricted distribution of NMU receptors to a region of the spinal cord involved in nociception suggests that this peptide receptor system may play a role in nociception.

A pro-nociceptive role of neuromedin U in adult mice.

Although the neuropeptide neuromedin U (NMU) was first isolated from the spinal cord, its actions in this site are unknown. The recent identification of the NMU receptor subtype 2 (NMU2R) in the spinal cord has increased the interest in investigating the role of NMU in this part of the central nervous system. Here, we report a novel function for NMU in spinal nociception in the mouse. Systemic perfusion of NMU (rat, NMU‐23) dose‐dependently (0.2, 0.5, 1, and 2.5 &mgr;M) potentiated both the background activity and noxious pinch‐evoked response of nociceptive or wide dynamic range, but not non‐nociceptive, dorsal horn neurons. At 2.5 &mgr;M, NMU‐23 increased the total background activity from 154±34 to 1374±260 spikes/160 s (P<0.005, n=28) and increased the evoked nociceptive response by 185±50% (P<0.01, n=13). Intrathecal administration of NMU‐23 (0.4, 1.1, and 3.8 nmol/10 &mgr;l) dose‐dependently decreased thermal withdrawal latencies and produced a morphine‐sensitive behavioral response. These electrophysiological and behavioral results suggest that NMU may be a novel physiological regulator in spinal nociceptive transmission and processing.

Differential expression and pharmacology of native P2X receptors in rat and primate sensory neurons.

Evidence suggesting the involvement of P2X2 and P2X3 in chronic pain has been obtained mostly from rodent models. Here we show that rodents may be poor predictors of P2X3 pharmacology in human. We demonstrate that monkey and human dorsal root ganglion (DRG) neurons do not express appreciable levels of P2X2 subunit, contrary to rat sensory neurons. Additionally, we report functional P2X3 activity in monkey DRG neurons and confirm the absence of functional P2X2/3 receptors. Interestingly, native P2X3 receptors in rat and monkey DRGs show similar agonist potency, but different antagonist potencies for TNP-ATP [2-O-(2,4,6-trinitrophenyl)-ATP] and RO51. This unexpected difference in antagonist potency was confirmed by comparing rat and human P2X3 receptors in HEK293 cells. Mutagenesis studies reveal that two extracellular residues, A197 and T202, are synergistically responsible for the potency drop in primate P2X3 receptors. These results uncover species-specific P2X3 pharmacology and identify key mechanisms impacting the translatability of potential analgesics targeting P2X3 receptors.

Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) hydrolyzes several bioactive lipids including the endocannabinoid anandamide. Synthetic FAAH inhibitors are being generated to help define the biological role(s) of this enzyme, the lipids it degrades in vivo, and the disease states that might benefit from its pharmacological modulation. AZ513 inhibits human FAAH (IC(50)=551 nM), is 20-fold more potent against rat FAAH (IC(50)=27 nM), and is inactive at 10 μM against the serine hydrolases acetylcholinesterase, thrombin, and trypsin. In contrast to most other potent FAAH inhibitors, AZ513 showed no evidence of covalently modifying the enzyme and displayed reversible inhibition. In an enzyme cross-competition assay, AZ513 did not compete with OL-135, an inhibitor that binds to the catalytic site in FAAH, which indicates that AZ513 does not bind to the catalytic site and is therefore noncompetitive with respect to substrate. AZ513 has good cell penetration as demonstrated by inhibition of anandamide hydrolysis in human FAAH-transfected HEK293 cells (IC(50)=360 nM). AZ513 was tested in a rat spinal cord slice preparation where CB(1) activation reduces excitatory post-synaptic currents (EPSCs). In this native tissue assay of synaptic activity, AZ513 reduced EPSCs, which is consistent with inhibiting endogenous FAAH and augmenting endocannabinoid tone. AZ513 has a unique biochemical profile compared with other published FAAH inhibitors and will be a useful tool compound to further explore the role of FAAH in various biological processes.

Functional genomics of the rat neuromedin U receptor 1 reveals a naturally occurring deleterious allele

Neuromedin U (NMU) plays an important role in a number of physiological processes, but the relative contribution of its two known receptors, NMUR1 and NMUR2, is still poorly understood. Here we report the existence of a SNP T(1022)→A (Val(341)→Glu) in the third exon of the rat Nmur1 gene that leads to an inactive receptor. This SNP is present within the coding region of the highly conserved NPXXY motif found within all class A type G protein-coupled receptors and translates to an NMUR1 receptor that is not expressed on the cell surface. Genetic analysis of the Nmur1 gene in a population of Sprague-Dawley rats revealed that this strain is highly heterogeneous for the inactivating polymorphism. The loss of functional NMUR1 receptors in Sprague-Dawley rats homozygous for the inactive allele was confirmed by radioligand binding studies on native tissue expressing NMUR1. The physiological relevance of this functional genomics finding was examined in two nociceptive response models. The pronociceptive effects of NMU were abolished in rats lacking functional NMUR1 receptors. The existence of naturally occurring NMUR1-deficient rats provides a novel and powerful tool to investigate the physiological role of NMU and its receptors. Furthermore, it highlights the importance of verifying the NMUR1 single nucleotide polymorphism status for rats used in physiological, pharmacological or toxicological studies conducted with NMUR1 modulators.